This invention relates to a novel method for chromatographically separating PTH-amino acids (i.e., 3-phenyl-2-thiohydantoin derivatives of amino acids) by changing the hydrophilic/hydrophobic balance on the stationary phase surface in an aqueous system by an external signal (for example, temperature).
Amino acid sequences of proteins, peptides, etc. are determined by the Edman degradation method which comprises successively, from the terminal, liberating amino acid residues each as anilinothiazolinone, and then converting it into a stable phenylthiohydantoin derivative (PTH-amino acid) under acidic conditions followed by identification. In the identification, it has been a practice to use liquid chromatography with the use of a reversed phase column. By using the reversed phase column, all of 20 amino acid species constituting proteins can be separated from each other by a single analytical operation and analyzed within 1 hour at a relatively high sensitivity.
There are marketed various reversed phase columns wherein porous silica gel is employed as the stationary phase. On the silica gel, octadecylsilane group, phenyl group, etc. are fixed by chemical bonds thereby making the surface of the silica carrier hydrophobic. On the surface of this reversed phase, various PTH-amino acids are separated from each other due to the difference in the degree of hydrophobicity. As the chromatographic eluting solution, use is made of a mixture of a buffer solution with an organic solvent miscible with water.
PTH-amino acids may be eluted by continuously increasing the organic solvent concentration in the mobile phase. Alternatively, it may be eluted at a constant organic solvent concentration. In either case, elution is performed by changing the hydrophobicity or hydrophilicity of the eluting solvent. As the buffer solution, sodium acetate-based ones and ammonium acetate-based ones are mainly employed. As the organic solvent, acetonitrile and methanol are mainly employed. The separating ability widely varies depending on various separation conditions such as the flow rate of the elution solvent, ionic strength, pH, column temperature, etc. In general, separation at a higher temperature can give the more favorable results, though the performance varies from column to column. The PTH-amino acids thus separated are detected with an UV detector at a wavelength of, for example, 254 nm or 268 nm and then identified by comparing in elution time with standard PTH-amino acids.
However, the organic solvents and buffer solutions employed as the conventional mobile phases contaminate the eluates and bring about extremely strong UV absorption. Thus, there arises a problem that the stability of a base line and sensitivity are seriously lowered thereby. Moreover, the column should be washed and equilibrated with the initial eluting solution before starting continuous analysis, which brings about another problem, i.e., a prolonged analysis time. In addition, it is feared that these organic solvents and buffer solutions would induce environmental pollution. Accordingly, it has been required to establish a separation method without resort to these substances.
To solve the above-described problem, JP (Kokai) Hei 7-318551 proposes a chromatographic method with the use of a chromatographic packing capable of separating or purifying biological factors (proteins, DNAs, saccharides, lipids, etc.) or cells by regulating the interaction of these substance with the surface of a solid in an aqueous system by an external signal (for example, temperature). By using this method, biological factors or cells can be separated or purified by regulating the surface characteristics of the stationary phase under a temperature change while fixing the mobile phase to an aqueous system without resort to any organic solvent or buffer solution as the mobile phase. Thus, the biological factors (proteins, etc.) or cells can be separated and collected while sustaining the functions thereof in a single aqueous mobile phase, thus preventing the contamination with impurities.
However, the solutes which can be separated or purified by the method reported by JP (Kokai) Hei 7-318551 are physiologically active proteins, cells and the like. More particularly, the above patent discloses exclusively bovine serum albumin, IgG, fibrinogen, fibronectin, transferrin, blood coagulation factor, etc. (see, for example, page 5, column 7, lines 5-10 in the official gazette thereof). Namely, no other substance is stated therein.
To develop a method for analyzing PTH-amino acids within a short time at a high sensitivity, the present inventors have conducted intensive studies. As a result, they have found a fact, as an unexpected turn even for a person skilled in the art, that PTH-amino acids can be analyzed by using the method described in JP (Kokai) Hei 7-318551, though free amino acids cannot be analyzed thereby. The present invention has been completed based on this finding.
Accordingly, the present invention provides a method for separating PTH-amino acids characterized by chromatographically separating said PTH-amino acids with the use of a packing wherein the hydrophilic/hydrophobic balance on the stationary phase surface can be changed by an external signal while fixing the mobile phase to an aqueous system.
The present invention further provides a method for separating PTH-amino acids characterized by comprising retaining said PTH-amino acids by a stationary phase comprising a chromatographic packing chemically modified with a polyalkylacrylamide having terminal amino, carboxyl, hydroxyl groups, etc. or a copolymer of the same; and allowing the PTH-amino acids to pass through a single mobile phase while changing the hydrophilic/hydrophobic balance on the stationary phase surface by the temperature gradient method wherein the external temperature is changed stepwise to thereby separate the PTH-amino acids.